Reduction in the radiation-induced late S phase and G2 blocks in HL-60 cell populations by amiloride, an efficient inhibitor of the Na+/H+ transporter.
Recent investigations that showed that amiloride delayed or inhibited apoptosis indicated it might also attenuate cell cycle checkpoints activated by ionizing radiation. In this report, single- and dual-parameter flow cytometry were used to investigate the effects of amiloride on cell cycle progression, and the effectiveness of amiloride to attenuate the S and G2 phase checkpoint responses induced by 2.5, 5.0, and 7.5 Gy of gamma radiation. The late S-phase delay, noted at 8 h following irradiation, and a radiation-induced G2 block, which was maximum at 16 h after irradiation, were both significantly reduced in amiloride-treated samples. Attenuation of the radiation-induced late S phase and G2 blocks resulted in cell division without apparent apoptosis or necrosis over a 24-h period. Results presented indicate that amiloride reduces the radiation-induced G2 block in HL-60 cell populations almost equally well as caffeine and to a greater extent than staurosporine. Immunofluorescent detection and quantitation of cyclin B1 expression demonstrated that amiloride only significantly reduced cyclin B1 expression following 5.0 Gy, when there was a notable induction of a significant G2 delay, followed by a relatively rapid recovery in cycling potential. The results suggest that amiloride affects the radiation-triggered signaling cascades to alter the kinase activity of proteins associated with mitotic progression, particularly the cyclin B1-p34cdc2 complex. Alternatively, alterations in intracellular ion concentrations induced by amiloride may lead to changes in Ca2+-dependent signaling cascades and thereby decrease the radiation-mediated cell cycle perturbations.
Sailer
B L
BL
Los Alamos National Laboratory, Life Sciences Division, New Mexico 87545, USA.
Barrasso
A M
AM
Valdez
J G
JG
Cobo
J M
JM
D'Anna
J A
JA
Crissman
H A
HA
D000584:Amiloride / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002110:Caffeine / Q000494:pharmacology; D019926:Cyclin B / Q000096:biosynthesis / Q000235:genetics; D056744:Cyclin B1; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects* / Q000528:radiation effects; D005720:Gamma Rays; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects / Q000528:radiation effects; D018922:HL-60 Cells / Q000187:drug effects* / Q000528:radiation effects; D006801:Humans; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects* / Q000528:radiation effects; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*; D019311:Staurosporine / Q000494:pharmacology
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D011837:Radiation-Protective Agents; D017923:Sodium-Hydrogen Exchangers; D002110:Caffeine; D000584:Amiloride; D019311:Staurosporine